甲肝病毒与MicroRNA-122光化学传感器的构建及其研究

发布时间：2018-04-24 18:18

本文选题：分子印迹 + SiO_2@MPS-CdTe/CdS　；参考：《湘潭大学》2017年硕士论文

【摘要】：光化学生物传感器是对生物体及活细胞中生物分子实时分析检测的重要工具。光化学生物传感器具有多种内在优点,如灵敏度高、仪器简单、高通量的筛选能力。本论文基于分子印迹技术、链置换循环扩增技术构建了三种不同的光化学生物传感器,并用于对生物分子甲肝病毒HAV及MicroRNA-122的高灵敏度高选择性检测。主要研究工作如下:(1)温敏型甲肝病毒分子印迹共振光传感器的构建及研究通过温敏印迹聚合物包覆SiO_2构建了一个共振光病毒分子印迹传感器高选择性检测HAV。该传感器以N-异丙基丙烯酰胺作为温度敏感单体,对病毒的特异性识别性能可以通过温度调控,在40°C时特异性捕获病毒引起分子印迹聚合物共振光增强,20°C时释放病毒共振光减弱。此外,传感器的响应时间为30 min,检出限为1.1 pM。该传感器成功应用于稀释人体血清中HAV的检测。三个不同浓度的HAV回收率为90.8%-108.3%。该传感器为病毒高灵敏度高选择性检测提供了一个快速有效的方法。(2)SiO_2@MPS-CdTe/CdS MIP荧光传感器的构建及对甲肝病毒的检测通过溶胶-凝胶聚合反应,使用甲肝病毒作为模板合成了一个新型SiO_2@MPS-CdTe/CdS@MIPs荧光传感器用于特异性识别检测甲肝病毒(HAV)。当HAV存在时,CdTe/CdS的能量转移给HAV造成荧光淬灭。传感器的荧光强度在20 min内显著下降,检出限为88 pM。该传感器成功应用于稀释200倍人体血清中HAV的检测,三个不同浓度HAV的回收率为96.7%-103.8%。该传感器克服了分子印迹病毒传感器响应时间长的问题,为病毒检测提供了一个快速有效的方法。(3)基于链扩增技术的比率型荧光传感器对miRNA-122的检测通过利用2-氨基嘌呤(2-AP)和硫黄素T(ThT)作信号源,构建了一个新型的基于杂交链反应的比率型荧光传感器检测miRNA-122。随着mi RNA-122浓度的增加,发夹DNA序列H1环部修饰的2-AP经链置换反应至双链DNA中,2-AP的荧光淬灭;同时由于H1打开,K+稳定的G-四联体形成,ThT荧光增强。该荧光传感器对miRNA-122的检测表现出高的选择性及低的检出限72 pM;响应时间为80 min。此外,该传感器成功应用于人体癌细胞提取液中mi RNA-122的检测。这个简单、免酶切的比率型荧光传感器不仅丰富了microRNAs传感器的研究,而且在miRNA相关生物化学研究及临床诊断具有潜在的应用。
[Abstract]:Photochemical biosensor is an important tool for real-time analysis and detection of biomolecules in organisms and living cells. Photochemical biosensor has many inherent advantages, such as high sensitivity, simple instrument and high throughput screening ability. In this paper, three different photochemical biosensors were constructed based on molecular imprinting technique and chain displacement cyclic amplification technique, and they were used to detect HAV and MicroRNA-122 with high sensitivity and high selectivity. The main work of this paper is as follows: (1) the construction of thermo-sensitive molecular imprinting resonance light sensor for hepatitis A virus and the construction of a highly selective SiO_2 sensor based on thermo-imprinted polymer coated with SiO_2. The sensor uses N- isopropylacrylamide as a temperature sensitive monomer, and the specific recognition performance of the virus can be controlled by temperature. The specific viral capture at 40 掳C caused a decrease in the resonant light intensity of the molecular imprinted polymer at 20 掳C. In addition, the response time of the sensor is 30 min and the detection limit is 1.1 pm. The sensor has been successfully applied to the detection of HAV in diluted human serum. The recoveries of three different concentrations of HAV ranged from 90.8 to 108.3. The sensor provides a rapid and effective method for the high sensitivity and high selectivity of virus detection. The construction of MPS-CdTe / CDs MIP fluorescent sensor and the detection of hepatitis A virus by sol-gel polymerization. A novel SiO_2@MPS-CdTe/CdS@MIPs fluorescence sensor was synthesized using hepatitis A virus as a template for specific identification and detection of hepatitis A virus (HAV). The energy transfer of CdTe / CDs in the presence of HAV results in fluorescence quenching of HAV. The fluorescence intensity of the sensor decreased significantly within 20 min, and the detection limit was 88 pm. The sensor has been successfully applied to the detection of HAV in human serum of 200 times dilution. The recoveries of three different concentrations of HAV are 96.7- 103.8. The sensor overcomes the problem of long response time of the molecularly imprinted virus sensor. To provide a rapid and effective method for virus detection, a ratio fluorescence sensor based on chain amplification technique was used to detect miRNA-122 by using 2-Aminopurine 2-AP) and thiocyanin Thytht as signal sources. A novel ratio fluorescence sensor based on hybrid chain reaction was constructed to detect miRNA-122. With the increase of the concentration of mi RNA-122, the H1 ring modified 2-AP of hairpin DNA sequence was quenched by chain replacement reaction to the fluorescence of 2-AP in double-stranded DNA. The fluorescence sensor showed high selectivity and low detection limit of 72 pm for miRNA-122 detection, and the response time was 80 min. In addition, the sensor has been successfully applied to the detection of mi RNA-122 in human cancer cell extract. This simple, digested ratio fluorescent sensor not only enriches the research of microRNAs sensors, but also has potential applications in the biochemical research and clinical diagnosis of miRNA.
【学位授予单位】：湘潭大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373;O657.3

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